1. Field of the Invention
The present invention relates to a mass analysis method and a mass analysis apparatus for conducting analysis on proteins or peptides in samples by mass analysis.
2. Background Art
Proteins in vivo play a number of roles such as formation of cytoskeleton, muscle contraction, host defense (immunization), signal transduction, various types of catalysis or blood coagulation. However, the specific role of each protein has not been elucidated for many of the proteins.
Thus, while focusing on presence/absence of a particular disease, presence/absence of an administration of a drug, temporal changes of medication, presence/absence or degree of physical stress, and the difference in tissues, attempts have been made to comprehensively detect proteins expressed in vivo and detect the difference of proteins which characterizes them.
For example, if a protein that is expressed in patients suffering from a particular disease but not expressed in healthy persons is found, the protein may be associated with the disease in some way or other. Further, research on functions of the protein may clarify a cause of the disease and enable the development of a drug. Also, since a characteristic to the disease is given, there is a possibility that accurate diagnosis will be available among a group of diseases giving the similar findings.
In this way, it is considered very important to comprehensively identify and comparatively analyze proteins in vivo.
For this purpose, known is a method of analyzing a protein with a mass analysis apparatus. In this analysis method, an ion derived from a peptide molecule is selected as a precursor ion, and fragmented and then, mass spectrum having a structural data of the peptide (that is, MS/MS spectrum or MS2 spectrum) is obtained, which are realized.
In mass analyzing successively components separated by liquid chromatograph, MS1 spectrum (mass spectrum obtained by observing a precursor ion in contrast to MS2 spectrum) includes time series information derived from each component separated by liquid chromatograph. In particular, when a peptide is ionized by ESI (Electrospray Ionization), a proton (hydrogen ion: H+) or the like is added to or eliminated from its molecular ion, which is often observed as a multiply-charged ion.
Further, MS2 spectrum includes structural information regarding a precursor ion. Particularly, in the case of peptide, for example, an ion generated by fragmentation of C—N bond in peptide bond of amino acid is observed, and structural knowledge is often obtained. As long as a precursor ion providing MS2 spectrum is derived from peptide separated by liquid chromatograph, the same precursor ion is ideally observed at the same retention time when the measurement is carried out under the same conditions.
Furthermore, a method is realized wherein a precursor ion is selected from ions observed in MS2 spectrum and fragmented for the obtainment of mass spectrum (that is, MS3 spectrum). In particular, when MS2 spectra are very similar even though ions have different structures, or when the number of types of ions observed in MS2 spectrum is small and structural information is poor, the obtainment of MS3 spectrum is sometimes effective in structure analysis.
Patent Document 1 describes sample evaluation by mass spectrum comparison. In this patent document 1, a standard signal of a fragment of a viral gene transfer vector is prepared, a sample signal is compared with the standard signal, the presence of impurities is detected, and a signal corresponding to an impurity fragment is identified.
[Patent Document 1] JP Patent Publication (Kohyo) No. 2003-510008
However, the above conventional art has the following problems in identifying a protein and peptide in sample.
(a) The Number of Identifications of a Plurality of Components in Sample
In conducting identification with mass spectrum of a precursor ion derived from components in a sample, ions from all components are selected as precursor ions, and it is ideal to obtain mass spectra thereof However, there are restrictions on time during which candidate ions to a precursor ion are observable or measurement time necessary for obtaining MS/MS spectra. Thus, it is not necessarily possible to select ions corresponding to all the existing components as precursor ion and to obtain MS/MS spectra thereof
In other words, if ions derived from all the components are selected as precursor ions to obtain mass spectra thereof, it requires very long time. Therefore, it is difficult for practical purpose.
(b) Measurement Time and Sample Amount
As a method to increase the above number of identifications, a method is provided wherein the same sample is measured several times and information regarding more components is obtained. However, repetition of the measurement needs more time, resulting in decreased throughput. Further, an amount of the sample should be enough to conduct several-times measurement, so it is difficult to conduct measurement when the sample availability is low.
(c) Time Necessary for Component Identification
Even when MS/MS spectra are obtained from many kinds of ions, it requires time to analyze corresponding components, so it is not necessarily efficient. In particular, in a method of separating protein or peptide by electrophoresis, electrophoresis itself consumes time.
(d) Identification of Trace Component
In the case of electrophoresis, a certain concentration is required for image identification, and thus less amount of trace component is neglected. Further, in analyzing an enzymatically digested sample by liquid chromatograph, MS/MS spectra derived from trace components having close retention times cannot often be obtained.
In particular, when measurements and analyses are repeated on plural kinds of samples, the above points are more important.